[0001] The present invention relates to a certain 3′-fluoro nucleoside analogue, pharmaceutically
acceptable derivatives thereof, and the use of such compounds in therapy, particularly
for the treatment or prophylaxis of certain viral infections.
[0002] One group of viruses which has recently assumed a particular importance are the retroviruses.
Retroviruses form a sub-group of RNA viruses which, in order to replicate, must first
'reverse transcribe' the RNA of their genome into DNA ('transcription' conventionally
describes the synthesis of RNA from DNA). Once in the form of DNA, the viral genome
may be incorporated into the host cell genome, allowing it to take advantage of the
host cell's transcription/translation machinery for the purposes of replication. Once
incorporated, the viral DNA is virtually indistinguishable from the host's DNA and,
in this state, the virus may persist for the life of the cell.
[0003] A species of retrovirus, Human Immunodeficiency Virus (HIV), has been reproducibly
isolated from patients with Acquired Immune Deficiency Syndrome (AIDS) or with the
symptoms that frequently precede AIDS. AIDS is an immunosuppressive or immunodestructive
disease that predisposes subjects to fatal opportunistic infections. Characteristically,
AIDS is associated with a progressive depletion of T-cells, especially the helper-inducer
subset bearing the OKT⁴ surface marker. HIV is cytopathic and appears to preferentially
infect and destroy T-cells bearing the OKT⁴ marker and it is now generally recognised
that HIV is the etiological agent of AIDS.
[0004] Since the discovery that HIV is the etiological agent of AIDS, numerous proposals
have been made for anti-HIV chemotherapeutic agents that may be effective in treating
AIDS. Thus, for example, European Patent Specification No. 196185 describes 3′-azido-3′-deoxythymidine
(which has the approved name zidovudine), its pharmaceutically acceptable derivatives
and their use in the treatment of human retrovirus infections including AIDS and associated
clinical conditions. Other nucleoside derivatives that have been suggested for the
treatment of HIV infections include the 3′-fluoronucleosides described for example
in European Patent Specification 254 268 and International Patent Specification 88/0050.
[0005] Another group of viral pathogens of major consequence worldwide are the hepatitis
viruses, in particular hepatitis B virus (HBV). HBV is most common in Asian countries,
and prevalent in sub-Saharan Africa. The virus is etiologically associated with primary
hepatocellular carcinoma and is thought to cause 80% of the world's liver cancer.
In the United States more than ten thousand people are hospitalised for HBV illness
each year, and an average of 250 die with fulminant disease. The United States currently
contains an estimated pool of 500,000-1 million infectious carriers. Chronic active
hepatitis will develop in over 25% of carriers, and often progresses to cirrhosis.
It is estimated that 5000 people die from HBV related cirrhosis each year in the USA,
and that perhaps 1000 die from HBV-related liver cancer. Thus, there is a great need
for effective antiviral agents, both to control the chronic infection and reduce progression
to hepatocellular carcinoma.
[0006] Clinical effects of infection with HBV range from headache, fever, malaise, nausea,
vomiting, anorexia and abdominal pains. Replication of the virus is usually controlled
by the immune response, with a course of recovery lasting weeks or months in humans,
but infection may be more severe leading to persistent chronic liver disease as outlined
above. In "Viral Infections of Humans" (second edition, Ed., Evans, A.S. (1982) Plenum
Publishing Corporation, New York), Chapter 12 describes the etiology of viral hepatitis
infections.
[0007] We have now surprisingly discovered that 2′,3′-dideoxy-5-ethynyl-3′-fluorouridine
as referred to below has potent activity against retroviruses such as HIV, as well
as HBV.
[0008] According to the present invention therefore we provide the compound of formula (I):
also characterised by the name 2′,3′-dideoxy-5-ethynyl-3′-fluorouridine; and pharmaceutically
acceptable derivatives thereof. Hereinafter the compound of formula (I) and its pharmaceutically
acceptable derivatives will be referred to as compounds according to the invention.
[0009] Formula (I) above depicts the compound in the keto tautomeric form. It will be appreciated
that the compound may also exist in the corresponding enol tautomeric form.
[0010] In a further aspect of the invention there are provided the compounds according to
the invention for use in medical therapy particularly for the treatment or prophylaxis
of viral infections especially retroviral infections and hepatitis B viral infections.
[0011] Examples of retroviral infections which may be treated or prevented in accordance
with the invention include human retroviral infections such as HIV-I, HIV-2 and Human
T-cell Lymphotropic Virus (HLTV) e.g. HTLV-I or HTLV-II infections.
[0012] The compounds according to the invention are also useful for the treatment or prophylaxis
of clinical conditions associated with retroviral infections, for example, AIDS, Kaposi's
sarcoma, thrombocytopenia purpura, AIDS-related complex (ARC), progressive generalized
lymphadenopathy (PGL), and patients carrying AIDS-antibodies or who are seropositive
to the HIV virus, as well as chronic neurological conditions such as multiple sclerosis
or tropical spastic paraparesis.
[0013] The compounds according to the invention may also be used for the treatment or prophylaxis
of infections carried by DNA viruses which, like retroviruses, are incorporated into
the host genome during their life-cycle, i.e. DNA viruses such as hepatitis B. Thus,
there is further provided the compounds according to the invention for use in the
treatment or prophylaxis of infections caused by such retrovirus-like viruses.
[0014] In a further aspect of the present invention there is included:-
a) A method for the treatment or prophylaxis of a viral infection of a mammal including
man which comprises treating the mammal with an antivirally effective amount of a
compound according to the invention.
b) Use of a compound according to the invention in the manufacture of a medicament
for the treatment or prophylaxis of any of the above-mentioned infections or indications.
[0015] By "a pharmaceutically acceptable derivative" is meant any pharmaceutically acceptable
salt, ester, or salt of such ester, of the compound of formula (I) or any other compound
which, upon administration to the recipient, is capable of providing (directly or
indirectly) such a compound or an antivirally active metabolite or residue thereof.
[0016] Preferred esters of the compound of formula (I) include carboxylic acid esters in
which the non-carbonyl moiety of the ester grouping is selected from straight or branched
chain alkyl (e.g. methyl, n-propyl, n-butyl or t-butyl), alkoxyalkyl (e.g. methoxymethyl),
aralkyl (e.g. benzyl), aryloxyalkyl (e.g. phenoxymethyl), aryl (e.g. phenyl optionally
substituted by halogen, C₁₋₄ alkyl or C₁₋₄ alkoxy or amino); sulphonate esters such
as alkyl- or aralkylsulphonyl (e.g. methanesulphonyl); amino acid esters (e.g. L-valyl
or L-isoleucyl); and mono-, di- or tri-phosphate esters. In such esters unless otherwise
specified, any alkyl moiety present advantageously contains 1 to 18 carbon atoms,
particularly 1 to 4 carbon atoms. Any aryl moiety present in such esters advantageously
comprises a phenyl group. Any reference to any of the above compounds also includes
a reference to a pharmaceutically acceptable salt thereof.
[0017] Examples of pharmaceutically acceptable salts of the compound of formula (I) and
pharmaceutically acceptable derivatives thereof include base salts, e.g. derived an
appropriate base, such as alkali metal (e.g. sodium), alkaline earth metal (e.g. magnesium)
salts, ammonium and NX⁺₄ (wherein X is C₁₋₄ alkyl).
[0018] The compounds according to the invention may be employed in combination with other
therapeutic agents for the treatment or prophylaxis of the above infections or conditions.
Examples of such further therapeutic agents include agents that are effective for
the treatment or prophylaxis of HIV infections or associated conditions such as 3′-azido-3′-deoxythymidine
(zidovudine), other 2′,3′-dideoxynucleosides such as 2′,3′-dideoxycytidine, 2′,3′-dideoxyadenosine
and 2′,3′-dideoxyinosine, carbovir, acyclic nucleosides (e.g. acyclovir), 2′,3′-didehydrothymidine,
interferons such as α-interferon, renal excretion inhibitors such as probenicid, nucleoside
transport inhibitors such as dipyridamole, as well as immunomodulators such as interleukin
II and granulocyte macrophage colony stimulating factors, phosphonoformic acid and
soluble CD₄ and genetically engineered derivatives thereof. The component compounds
of such combination therapy may be administered simultaneously, in either separate
or combined formulations, or at different times, e.g. sequentially, such that a combined
effect is achieved.
[0019] The compounds according to the invention, also referred to herein as active ingredients,
may be administered for therapy by any suitable route including oral, rectal, nasal,
topical (including buccal and sublingual), vaginal and parenteral (including subcutaneous,
intramuscular, intravenous and intradermal. It will also be appreciated that the preferred
route will vary with the condition and age of the recipient, the nature of the infection
and the chosen active ingredient.
[0020] In general a suitable dose will be in the range of 3.0 to 120 mg per kilogram of
body weight of the recipient per day, preferably in the range of 6 to 90 mg per kilogram
body weight per day and most preferably in the range 15 to 60 mg per kilogram body
weight per day. The desired dose is preferably presented as two, three, four, five,
six or more sub-doses administered at appropriate intervals throughout the day. These
sub-doses may be administered in unit dosage forms, for example, containing 10 to
1500 mg, preferably 20 to 1000 mg, and most preferably 50 to 700 mg of active ingredient
per unit dosage form.
[0021] Ideally, the active ingredient should be administered to achieve peak plasma concentrations
of the active compound of from about 1 to about 75 µM, preferably about 2 to 50 µM,
most preferably about 3 to about 30 µM. This may be achieved, for example, by the
intravenous injection of a 0.1 to 5% solution of the active ingredient, optionally
in saline, or orally administered as a bolus containing about 1 to about 100 mg/kg
of the active ingredient. Desirable blood levels may be maintained by a continuous
infusion to provide about 0.01 to about 5.0 mg/kg/hour or by intermittent infusions
containing about 0.4 to about 15 mg/kg of the active ingredient.
[0022] While it is possible for the active ingredient to be administered alone it is preferable
to present it as a pharmaceutical formulation. The formulations of the present invention
comprises at least one active ingredient, as defined above, together with one or more
acceptable carriers thereof and optionally other therapeutic agents. Each carrier
must be "acceptable" in the sense of being compatible with the other ingredients of
the formulation and not injurious to the patient. Formulations include those suitable
for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral
(including subcutaneous, intramuscular, intravenous and intradermal) administration.
The formulations may conveniently be presented in unit dosage form and may be prepared
by any methods well known in the art of pharmacy. Such methods include the step of
bringing into association the active ingredient with the carrier which constitutes
one or more accessory ingredients. In general, the formulations are prepared by uniformly
and intimately bringing into association the active ingredient with liquid carriers
or finely divided solid carriers or both, and then if necessary shaping the product.
[0023] Formulations of the present invention suitable for oral administration may be presented
as discrete units such as capsules, cachets or tablets each containing a predetermined
amount of the active ingredient; as a powder or granules; as a solution or suspension
in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil
liquid emulsion. The active ingredient may also be presented as a bolus, electuary
or paste.
[0024] A tablet may be made by compression or moulding, optionally with one or more accessory
ingredients. Compressed tablets may be prepared by compressing in a suitable machine
the active ingredient in a free-flowing form such as a powder or granules, optionally
mixed with a binder (e.g. povidone, gelatin, hydroxypropylmethyl cellulose), lubricant,
inert diluent, preservative, disintegrant (e.g. sodium starch glycollate, cross-linked
povidone, cross-linked sodium carboxmethyl cellulose) surface-active or dispersing
agent. Moulded tablets may be made by moulding in a suitable machine a mixture of
the powdered compound moistened with an inert liquid diluent. The tablets may optionally
be coated or scored and may be formulated so as to provide slow or controlled release
of the active ingredient therein using, for example hydroxypropylmethyl cellulose
in varying proportions to provide the desired release profile. Tablets may optionally
be provided with an enteric coating, to provide release in parts of the gut other
than the stomach.
[0025] Formulations suitable for topical administration in the mouth include lozenges comprising
the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth;
pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin,
or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable
liquid carrier.
[0026] Pharmaceutical compositions for topical administration according to the present invention
may be formulated as an ointment, cream, suspension, lotion, powder, solution, paste,
gel, spray, aerosol or oil. Alternatively, a formulation may comprise a dressing such
as a bandage or adhesive plaster impregnated with active ingredients and optionally
one or more excipients or diluents. Carriers which may be used include e.g. polyhydric
alcohols such as polyethylene glycols, propylene glycol or glycerol. Suitable excipients
are those known in the art to be appropriate.
[0027] Formulations for rectal administration may be presented as a suppository with a suitable
base comprising for example cocoa butter or a salicylate.
[0028] Formulations suitable for vaginal administration may be presented as pessaries, tampons,
creams, gels pastes, foams or spray formulations containing in addition to the active
ingredient such carriers as are known in the art to be appropriate.
[0029] Formulations suitable for parenteral administration include aqueous and non-aqueous
isotonic sterile injections solutions which may contain anti-oxidants, buffers, bacteriostats
and solutes which render the formulation isotonic with the blood of the intended recipient;
and aqueous and non-aqueous sterile suspensions which may include suspending agents
and thickening agents, and liposomes or other microparticulate systems which are designed
to target the compound to blood components or one or more organs. The formulations
may be presented in unit-dose or multi-dose sealed containers, for example, ampoules
and vials, and may be stored in a freeze dried (lyophilized) condition requiring only
the addition of the sterile liquid carrier, for example water for injections, immediately
prior to use. Extemporaneous injection solutions and suspensions may be prepared from
sterile powders, granules and tablets of the kind previously described.
[0030] Preferred unit dosage formulations are those containing a daily dose or unit, daily
sub-dose, as herein above recited, or an appropriate fraction thereof, of an active
ingredient.
[0031] It should be understood that in addition to the ingredients particularly mentioned
above the formulations of this invention may include other agents conventional in
the art having regard to the type of formulation in question, for example, those suitable
for oral administration may include such further agents as sweeteners, thickeners
and flavouring agents.
[0032] The compounds according to the invention may also be presented for the use in the
form of veterinary formulations, which may be prepared, for example, by methods that
are conventional in the art.
[0033] The present invention further includes a process for the preparation of the compound
of formula (I) and pharmaceutically acceptable derivaties thereof which comprises
either:
(A) removing a protecting group from a compound of formula (II):
(wherein X represents hydrogen or a hydroxy protecting group and Z represents hydrogen
or an ethynyl protecting group, providing at least one of X and Z represents a protecting
group);
(B) reacting a compound of formula (III):
(wherein Y represents a precursor group for the fluoro group) with an agent or under
conditions serving to convert the said precursor group to a fluoro group; or
(C) reacting a pyrimidine base of formula (IV):
or a functional equivalent thereof, with a compound serving to introduce the desired
ribofuranosyl ring at the 1-position of the pyrimidine base of formula (IV);
and thereafter, or simultaneously therewith, effecting one or more of the following
optional conversions:-
(i) removing any remaining protecting groups;
(ii) when a compound of formula (I) is formed, converting it into a pharmaceutically
acceptable derivative thereof;
(iii) when a pharmaceutically acceptable derivative of a compound of formula (I) is
formed, converting the said derivative into a compound of formula (I), or a different
derivative thereof.
[0034] In the above-described process according to the invention it will be appreciated
that the starting compounds of formulae (II), (III) and (IV), as well as the above-mentioned
agents and conditions, will be selected from those that are known in the art of nucleoside
synthetic chemistry. For example as described in Nucleic Acid Chemistry: Improved
New Synthetic Procedures, Methods and Techniques. Ed. L.B. Townsend and R.S. Tipson-Wiley
Interscience (1978) and Nucleoside Analogues: Chemistry, Biology and Medical Applications.
Ed. R.T. Walker, E. de Clercq and F. Eckstein, NATO Advanced Study Instituted, Plenum
press (1979). Examples of such conversion procedures are described hereinafter for
guidance and it will be understood that they can be modified in conventional manner
depending on the desired compound of formula (I). In particular, where a conversion
is described which would otherwise result in the undesired reaction of labile groups
then such groups may be protected in conventional manner, with subsequent removal
of the protecting groups after completion of the conversion.
[0035] In process (A), X may represent for example a hydroxy protecting group e.g. an ester
grouping particularly C₁₋₆ alkanoyl (e.g. acetyl) or aroyl, (e.g. toluoyl), or an
alkoxycarbonyl (e.g. methoxycarbonyl); or an ether group such as a trialkylsilyl group,
e.g. t-butyldimethylsilyl or an aralkyl group e.g. triphenylmethyl. Such groups may
be converted for example by hydrolysis to the desired hydroxy group or, by transesterification,
of an ester group to an alternative ester group. A particularly preferred hydroxy
protecting group is the p-toluoyl group which may be removed for example by treatment
under basic conditions, e.g. with sodium methoxide/methanol, aqueous methylamine or
ammonia. The above toluoyl derivative may be prepared by treating the appropriate
parent compound with for example p-toluoyl chloride, in a base solvent such as pyridine.
[0036] Another preferred hydroxy protecting group is the acetyl group which may also be
removed under basic conditions, e.g. as described above. The acetyl derivative may
be prepared by treating the appropriate parent compound with for example, acetic anhydride
in pyridine.
[0037] Examples of the protecting groups of the ethynyl group represented by Z in formula
(II) include trialkylsilyl (e.g. trimethylsilyl) groups which may be removed by treatment
under basic conditions using for example sodium methoxide/methanol.
[0038] The compounds of formula (II) may be prepared for example by the method described
by Robins
et al, Can.J.Chem.
60, 554 et seq (1982), e.g. by treating a corresponding compound in which the 5-position
of the uracil base is substituted with a leaving group, for example halogen such as
iodine and in which the 5′-hydroxy group is protected for example by an acyl group
such as a p-toluoyl or acetyl group, with the appropriate protected alkynylene compound,
such as trimethylsilylacetylene, with a palladium catalyst and another catalyst such
as a copper (I) salt in the presence of an organic base, such as triethylamine, which
also serves as a solvent, at an elevated temperature such as 50
oC to give the protected 5-alkynyl nucleoside. A preferred palladium catalyst is bis(triphenylphosphine)
palladium dichloride and a preferred copper catalyst is cuprous iodide. The parent
compound can readily be obtained by removal of any alkynyl protecting groups for example
trialkylsilyl by treatment under basic conditions using for example sodium methoxide/methanol.
[0039] The starting material referred to above in which the 5-position of the uracil base
is substituted with a halogen (particularly a chlorine, bromine or iodine) atom may
be prepared for example by halogenating a corresponding uridine compound in which
the 5-position is unsubstituted and in which the 5′-hydroxy group is blocked, for
example by an acyl group such as p-toluoyl or acetyl group. Halogenation of the above
starting material may be effected in conventional manner, for example iodination using
iodine monochloride e.g. in methylene dichloride, or iodine in a solvent containing
nitric acid, bromination using bromine e.g. in glacial acetic acid, or chlorination
using a chlorine complex of iodobenzene, e.g. in glacial acetic acid.
[0040] The starting materials for the last-mentioned process, i.e. the 5′-hydroxy blocked
uracil nucleoside may be prepared as described for example by G. Kowollik
et al, J. Prakt. Chem. 1973. 315(5) 895-900 for the preparation of 2,′3′-dideoxy-3′-fluorouridine
and subsequent blocking of the 5′-hydroxy group in conventional manner, e.g. in the
case of acyl blocking groups, by treatment with an appropriate acyl halide (e.g. chloride)
or an anhydride as described above.
[0041] With regard to process (B), this may be effected for example by treatment of a compound
of formula III in which Y represents a leaving group e.g. hydroxy or protected hydroxy
such as mesyl or trifluorosulphonyl with an appropriate fluorinating agent such as
hydrogen fluoride, potassium fluoride, potassium hydrogen fluoride diethylaminosulphurtrifluoride
or tetra-n-butylammonium fluoride.
[0042] Process (C) may be effected for example by treating the pyrimidine base of formula
(IV) or a salt or protected derivative thereof, with 3′-deoxy-3′-fluorothymidine for
example in the presence of the appropriate pentosyl transferring enzyme or an organic
catalyst such as trimethylsilyl or trifluoromethane sulphonate in a buffered aqueous
solution.
[0043] The compound of formula (I) may be converted into a pharmaceutically acceptable ester
thereof by reaction with an appropriate esterifying agent, e.g. an acid halide or
anhydride. The compound of formula (I), including esters thereof, may be converted
into pharmaceutically acceptable salts thereof in conventional manner, e.g. by treatment
with an appropriate base. An ester or salt of a compound of formula (I) may be converted
into the parent compound, e.g. by hydrolysis.
[0044] The following Examples are intended for illustration only and are not intended to
limit the scope of the invention in any way. The term 'active ingredient' as used
in the Examples means a compound of formula (I) or a pharmaceutically acceptable derivative
thereof.
Example 1
a) 2′,3′-Dideoxy-3′-fluoro-5′-O-p-toluoyluridine
[0045] p-Toluoyl chloride (freshly distilled, 325mg, 2.10 mmol) was added to a solution
of 2′,3′-dideoxy-3′-fluorouridine (G. Kowollick
et al, J. Prakt.Chem. 315(5), 895,1973) (440mg, 1.91 mmol) in dry pyridine (10ml). The
solution was stirred at 50
o for 1.5 hour, and then at 25
o for 18 hours. The pyridine was evaporated and the residue dissolved in CHCl₃ (25ml).
This solution was extracted with 1M H₂SO₄ (5ml), then H₂O (2 x 10 ml), and dried MgSO₄.
Evaporation of CHCl₃ left a colourless glass (0.72g) which was chromatographed on
silica gel. Elution with 2% MeOH-CHCl₃ gave the title product as white solid foam.
b) 2′,3′-Dideoxy-3′-fluoro-5-iodo-5′-O-p-toluoyluridine
[0047] The product of Stage a) (200mg, 0.574 mmol), iodine monochloride (139 mg, 0.861 mmol),
and methylene chloride (10ml) were refluxed for 2 hours. The solution was decolourised
with a minimum of 2% aqueous NaHSO₃ (ca. 2ml). The aqueous layer was separated and
the organic layer washed with water (2x5ml) and dried (MgSO₄). Evaporation of the
solvent left a cream coloured solid foam identified as the title compound.
[0048] Yield = 0.25g, 92%.
c) 2′,3′-Dideoxy-3′-fluoro-5′-O-p-toluoyl-5-(trimethylsilylethynyl) uridine
[0049] The product of Stage b), (0.23 g, 0.485 mmol), cuprous iodide (10 mg), bis(triphenylphosphine)
palladium (II) chloride (10 mg), trimethylsilylacetylene (0.145 g, 1.455 mmol) and
dry triethylamine (15 ml) are stirred at 50
oC under a dry N₂ atmosphere for 3.0 hr. The cooled suspension is evaporated to dryness
and the dark residue taken up in dichloromethane (20 ml). The solution is washed successively
with 2% aqueous disodium ethylenediaminetetraacetic acid (2x30 ml), water 30 ml, dried
(MgSO₄) and evaporated to give the title compound which is recrystallized from ethanol.
d) 2′,3′-Dideoxy-5-ethynyl-3′-fluorouridine
[0050] A solution of the product of Stage c), in 0.2M sodium methoxide in methanol (freshly
prepared from sodium and methanol) is stirred at room temperature for 3.0 hr. then
neutralized by portionwise addition of Dowex 50 (H⁺) ion exchange resin. The resin
is filtered off and washed well with methanol. The filtrate is evaporated to dryness
and the residue partitioned between water and ether. The aqueous layer is washed with
ether then evaporated to dryness, the residue triturated with ethanol and the solid
filtered and washed with ether to give the title compound.
Example 2
a) 2′,3′-Dideoxy-3′-fluoro-5′-O-p-toluoyluridine
[0051] To a stirred solution of 2′,3′-dideoxy-3′-fluorouridine (1g, 4.34 mmoles) in dry
pyridine (25 ml) at 0
oC was slowly added freshly distilled p-toluoyl chloride (0.63ml, 4.78 mmoles). After
the addition was complete, the mixture was stirred at 50
oC for 1.5 hrs., cooled and the solvent removed under reduced pressure. The residue
was dissolved in chloroform (35 ml) and the solution extracted with 1M sulphuric acid
(2 x 20ml), water (2x30ml) and dried (sodium sulphate). Evaporation of the solvent
and purification of the residue by silica column chromatography eluting with 5% MeOH/CH₂Cl₂
afforded the title compound.
b) 2′,3′-Dideoxy-3′-fluoro-5-iodo-5′-O-p-toluoyluridine
[0053] A solution of the product of Stage a), (3g, 8.61 mmoles) and iodine monochloride
(2.1g 12.92 mmoles) in methylene chloride (60 ml) was heated at reflux for 2 hrs.
The cooled reaction mixture was diluted with methylene chloride (60 ml), washed with
the minimum quantity of 2% aqueous sodium sulphite solution to achieve decolorisation,
water (2x70ml) and dried (sodium sulphate). Evaporation of the solvent afforded the
title compound as a white foam.
c) 2′,3′-Dideoxy-3′-fluoro-5′-O-p-toluoyl-5-(trimethylsilylethynyl) uridine
[0055] A solution of the product of Stage b), (0.8g, 1.69 mmoles), bis(triphenylphosphine)
palladium (II) chloride (25 mg) and copper (I) iodide (25 mg) in dry triethylamine
(40ml) and N,N-dimethylformamide (3ml) was degassed thoroughly with nitrogen. (Trimethylsilyl)
acetylene (0.47ml, 3.37 mmoles) was added and the mixture stirred under N₂ at 50
o for 8 hours. The solvent was removed under reduced pressure, the residue dissolved
in methylene chloride (40ml) and the solution washed with 2% aqueous disodium EDTA
solution (4ml), water (50ml) and dried (sodium sulphate). Evaporation of the solvent
and purification of the residue by silica column chromatography eluting with 2% MeOH/CH₂Cl₂
afforded the title compound. Trituration with ether/hexane afforded analytically pure
title compound as an off-white powder.
[0056] Yield: 0.56g, 74%
M.pt. = 130
oC
Microanalysis: |
calculated |
C,59.49; |
H,5.63 ; |
N,6.30% |
|
found |
C,59.54; |
H,5.75 ; |
N,6.29% |
d) 2′,3′-Dideoxy-5-ethynyl-3′-fluorouridine
[0057] The product of Stage c), (0.53g, 1.18mmoles) was dissolved in methanol (17ml) containing
sodium methoxide (from 0.027g, 1.18mmoles of sodium metal) and the solution left standing
at ambient temperature for 7 hours. The mixture was then neutralised with Dowex 50
(H⁺) resin, filtered and evaporated to dryness. The final residue was triturated with
ether (2 x 7 ml) and recrystallised from ethanol to give the title compound.
[0058] Yield = 0.144g, 50%
M.pt = 225 - 6
oC
Microanalysis: |
calculated |
C,51.99; |
H,4.33; |
N,11.02% |
|
found |
C,52.14; |
H,4.48; |
N,10.98% |
Example 3
a) 5′-O-Acetyl-2′,3′-dideoxy-3′-fluorouridine
[0059] Acetic anhydride (1.2ml, 13mmol) was added to a solution of 2′,3′-dideoxy-3′-fluorouridine
(1g, 4.34mmol) in dry pyridine (10ml) and the mixture was stirred at room temperature
for 24 hours. Ethanol (2ml) was added and the mixture was evaporated to dryness. Residual
pyridine was removed by coevaporation with portions of ethanol and the final residue
purified by silica gel column chromatography eluting with 5% MeOH/CH₂Cl₂ to give the
title compound which was isolated following trituration with ether.
b) 5′-O-Acetyl-2′,3′-dideoxy-3′-fluoro-5-iodouridine
[0061] Iodine monochloride (0.3ml, 6mmol) and the product of stage a) (0.91g, 3.34 mmol)
were combined in dichloromethane (10ml) and the mixture heated at reflux for 3 hours.
On cooling to room temperature, the solution was diluted with dichloromethane (20ml)
and washed with the minimum volume of 2% aqueous sodium sulphite solution to achieve
decolorisation, water (2x30ml) and dried (Na₂SO₄). Evaporation of the solvent afforded
the title compound as an off-white foam.
c) 5′-O-Acetyl-2′,3′-dideoxy-3′-fluoro-5-(trimethylsilylethynyl)uridine
[0063] A mixture of the product of stage b) (0.7g, 1.76mmol), bis-triphenylphosphine palladium
(II) chloride (0.036g) and copper (I) iodide (36mg) in redistilled triethylamine (35ml)
was degassed with oxygen-free nitrogen. Trimethylsilylacetylene (0.49ml, 3.52mmol)
was added and the mixture was stirred under a nitrogen atmosphere at room temperature
for 60 hours. The solvent was evaporated, the residue dissolved in dichloromethane
(30ml) and the solution washed with a 2% aqueous solution disodium EDTA (2x30ml),
water (40ml) and dried (Na₂SO₄). Evaporation of the solvent and purification of the
residue by silica gel column chromatography eluting with 40% ethyl acetate/toluene
afforded the title compound as a foam.
[0064] Yield = 0.37g, 58%
d) 2′,3′-Dideoxy-5-ethynyl-3′-fluorouridine
[0065] The product of stage c) (0.33g, 0.9mmol) was added to a solution of sodium methoxide
(from 0.021g, 0.9mmol of sodium metal) in dry methanol (8ml) and the mixture stirred
at room temperature for 5 hours. The solution was neutralised with Dowex 50 (H⁺) resin,
the resin filtered and washed with methanol (2x4ml) and the combined filtrate and
washings evaporated to dryness. The residue was washed with ether (2x5ml) and recrystallised
from acetonitrile to give pale yellow crystals of the title compound.
[0066] Yield = 0.17g, 74%
M.pt. 224-5
oC
Microanalysis |
calculated |
C,51.99; |
H,4.33; |
N,11.02% |
|
found |
C,51.87: |
H,4.40; |
N,10.90% |
Example 4
a) 2′,3′-Dideoxy-5-ethynyl-5′-O-(N-fluorenylmethoxy-carbonyl-L-isoleucinyl)-3′-fluorouridine
[0067] N,N′-Dicyclohexylcarbodiimide (0.57g, 2.8mmol) and N-fluoronylmethoxycarbonyl-L-isoleucine
(1g, 2.8mmol) were combined in dry methylene chloride (15ml) and the mixture was stirred
for 30 minutes at room temperature. The precipitated N,N′-dicyclohexylurea was filtered,
washed with methylene chloride (2 x 5 ml) and to the combined filtrate and washings
was added a solution of 2′,3′-dideoxy-5-ethynyl-3′-fluorouridine (0.3g, 1.18mmol)
and N,N-dimethylaminopyridine (0.087g, 0.72mmol) in dry dimethylformamide (5ml). The
mixture was stirred for 24 hours at room temperature and a further quantity of N,N′-dicyclohexylurea
was filtered. The filtrate was evaporated to dryness and the residue purified by column
chromatography eluting with 5%-15% acetone/methylene chloride to give a residue (0.64g)
which was further purified by addition of methylene chloride, filtration and evaporation
to give the title compound.
[0068] Yield = 0.57g, 82%
b) 2′,3′-Dideoxy-5-ethynyl-3′-fluoro-5′-O-L-isoleucinyluridine
[0069] A 20% solution of piperidine in dry dimethylformamide (5ml) was added to 2′,3′-dideoxy-5-ethynyl-5′-O-(N-fluorenylmethoxycarbonyl-L-isoleucinyl)-3′-fluorouridine
(0.57g, 0.96mmol) and after 4 minutes at room temperature, the solvents were evaporated
rapidly under high vacuum with minimal heating. Trituration of the residue with several
portions of ether afforded a crop of the title compound containing ∼5% of N,N′-dicyclohexylurea.
[0070] Yield = 0.145g, 41%
M.pt. 98-100
oC
Micronalysis |
calculated |
C,55.61; |
H,5.99; |
N,11.44% |
|
found |
C,55.70; |
H,6.32; |
N,11.10% |
Example 5
5′-O-Acetyl-2′,3′-dideoxy-5-ethynyl-3′-fluorouridine
[0071] To a stirred solution of 2′,3′-dideoxy-5-ethynyl-3′-fluorouridine (0.106g 0.4mmol)
in dry pyridine (5ml) at 0
oC was added acetic anhydride (0.05ml, 0.48mmol) and stirring maintained at 0
oC for 1.5 hours. After stirring at room temperature for 24 hours, a further aliquot
of acetic anhydride (0.02ml, 0.2mmol) was added and the mixture stirred at room temperature
for 3 hours. After quenching with methanol (1ml) the solvent was removed by evaporation
under reduced pressure and co-evaporated with portions of ethanol (2 x 30ml). The
residue was recrystallised from ethanol to give a white crystalline solid.
[0072] Yield = 0.079g, (64%)
M.pt. 160-161
oC
Microanalysis |
calculated |
C,52.70; |
H,4.392; |
N,9.46% |
|
found |
C,52.43; |
H,4.39; |
N,9.20% |
Example 6
2′,3′-Dideoxy-5-ethynyl-3′-fluoro-5′-O-(trimethylacetyl)uridine 0.2 hydrate
[0073] To a stirred solution of 2′,3′-dideoxy-5-ethynyl-3′-fluorouridine (0.106g 0.4mmol)
in dry pyridine (5ml) at 0
oC was added trimethylacetyl chloride (0.06ml, 0.48mmol) and stirring continued at
0
oC for 1.5 hours. After stirring at room temperature for 24 hours, a further aliquot
of the acid chloride (0.03ml, 0.24mmol) was added and stirring maintained for a further
3 hours. After quenching with methanol (3ml), the solvent was removed by evaporation
under reduced pressure and co-evaporated with several portions of ethanol (2 x 30ml)
and the resulting oil was chromatographed on a silica gel column eluting with 5% MeOH/CH₂Cl₂.
The appropriate fractions were combined and evaporated to dryness and the residue
was recrystallised twice from ethanol to give the chromatographically pure title product.
[0074] Yield = 0.063g, (45%)
M.pt. 182-185
oC
Microanalysis for 0.2 hydrate |
calculated |
C = 56.21; |
H = 5.68; |
N = 8.20% |
|
found |
C = 55.96; |
H - 5.57; |
N - 8.01% |
Example 7 Tablet Formulations
[0075] The following formulations A and B are prepared by wet granulation of the ingredients
with a solution of povidone, followed by addition of magnesium stearate and compression.
Formulation A |
|
mg/tablet |
mg/tablet |
(a) Active ingredient |
250 |
250 |
(b) Lactose B.P. |
210 |
26 |
(c) Povidone B.P. |
15 |
9 |
(d) Sodium Starch Glycollate |
20 |
12 |
(e) Magnesium Stearate |
5 |
3 |
|
500 |
300 |
Formulation B |
|
mg/tablet |
mg/tablet |
(a) Active ingredient |
250 |
250 |
(b) Lactose |
150 |
- |
(c) Avicel PH 101 |
60 |
26 |
(d) Povidone B.P. |
15 |
9 |
(e) Sodium Starch Glycollate |
20 |
12 |
(f) Magnesium Stearate |
5 |
3 |
|
500 |
300 |
Formulation C |
|
mg/tablet |
Active ingredient |
100 |
Lactose |
200 |
Starch |
50 |
Povidone |
5 |
Magnesium stearate |
4 |
|
359 |
[0076] The following formulations, D and E, are prepared by direct compression of the admixed
ingredients.
Formulation D |
|
mg/capsule |
Active Ingredient |
250 |
Pregelatinised Starch NF15 |
150 |
|
400 |
Formulation E |
|
mg/capsule |
Active Ingredient |
250 |
Lactose |
150 |
Avicel |
100 |
|
500 |
Formulation F (Controlled Release Formulation)
[0077] The formulation is prepared by wet granulation of the following ingredients with
a solution of povidone followed by the addition of magnesium stearate and compression.
|
mg/tablet |
(a) Active Ingredient |
500 |
(b) Hydroxypropylmethylcellulose (Methocel K4M Premium) |
112 |
(c) Lactose B.P. |
53 |
(d) Povidone B.P.C. |
28 |
(e) Magnesium Stearate |
7 |
|
700 |
[0078] Drug release takes place over a period of about 6-8 hours and is complete after 12
hours.
Example 8: Capsule Formulations
Formulation A
[0079] A capsule formulation is prepared by admixing the ingredients of Formulation D in
Example 4 above and filling into a two-part hard gelatin capsule.
Formulation B |
|
mg/capsule |
(a) Active ingredient |
250 |
(b) Lactose B.P. |
143 |
(c) Sodium Starch Glycollate |
25 |
(d) Magnesium Stearate |
2 |
|
420 |
[0080] Capsules are prepared by admixing the above ingredients and filling into a two-part
hard gelatin capsule.
Formulation C |
|
mg/capsule |
(a) Active ingredient |
250 |
(b) Macrogol 4000 BP |
350 |
|
600 |
[0081] Capsules are prepared by melting the Macrogol 4000 BP, dispersing the active ingredient
in the melt and filling the melt into a two-part hard gelatin capsule.
Formulation D |
|
mg/capsule |
Active ingredient |
250 |
Lecithin |
100 |
Arachis Oil |
100 |
|
450 |
[0082] Capsules are prepared by dispersing the active ingredient in the lecithin and arachis
oil and filling the dispersion into soft, elastic gelatin capsules.
Formulation E (Controlled Release Capsule)
[0083] The following controlled release capsule formulation is prepared by extruding ingredients
(a), (b) and (c) using an extruder, followed by spheronisation of the extrudate and
drying. The dried pellets are then coated with release- controlling membrane (d) and
filled into a two-piece, hard gelatin capsule.
|
mg/capsule |
(a) Active Ingredient |
250 |
(b) Microcrystalline Cellulose |
125 |
(c) Lactose BP |
125 |
(d) Ethyl Cellulose |
13 |
|
513 |
Example 9: Injectable Formulation |
Formulation A. |
Active ingredient |
0.200g |
Hydrochloric acid solution, 0.1M |
q.s. to pH 4.0 to 7.0 |
Sodium hydroxide solution, 0.1M |
q.s. to pH 4.0 to 7.0 |
Sterile water |
q.s. to 10ml |
[0084] The active ingredient is dissolved in most of the water (35
o-40
oC) and the pH adjusted to between 4.0 and 7.0 with the hydrochloric acid or the sodium
hydroxide as appropriate. The batch is then made up to volume with the water and filtered
through a sterile micropore filter into a sterile 10ml amber glass vial (type 1) and
sealed with sterile closures and overseals.
Formulation B. |
Active ingredient |
0.125 g |
Sterile, pyrogen-free, pH 7 phosphate buffer, q.s. to 25 ml |
Example 10: Intramuscular injection |
Active Ingredient |
0.20 g |
Benzyl Alcohol |
0.10 g |
Glycofurol 75 |
1.45 g |
Water for Injection q.s. to |
3.00 ml |
[0085] The active ingredient is dissolved in the glycofurol. The benzyl alcohol is then
added and dissolved, and water added to 3 ml. The mixture is then filtered through
a sterile micropore filter and sealed in sterile 3 ml amber glass vials (type 1).
Example 11: Syrup |
Active ingredient |
0.25 g |
Sorbitol Solution |
1.50 g |
Glycerol |
2.00 g |
Sodium Benzoate |
0.005 g |
Flavour, Peach 17.42.3169 |
0.0125 ml |
Purified Water q.s. to |
5.00 ml |
[0086] The active ingredient is dissolved in a mixture of the glycerol and most of the purified
water. An aqueous solution of the sodium benzoate is then added to the solution, followed
by addition of the sorbitol solution and finally the flavour. The volume is made up
with purified water and mixed well.
Example 12: Suppository |
|
mg/suppository |
Active Ingredient |
250 |
Hard Fat, BP (Witepsol H15 - Dynamit Nobel) |
1770 |
|
2020 |
[0087] One-fifth of the Witepsol H15 is melted in a steam-jacketed pan at 45
oC maximum. The active ingredient is sifted through a 200 µm sieve and added to the
molten base with mixing, using a silverson fitted with a cutting head, until a smooth
dispersion is achieved. Maintaining the mixture at 45°C, the remaining Witepsol H15
is added to the suspension and stirred to ensure a homogenous mix. The entire suspension
is passed through a 250 µm stainless steel screen and, with continuous stirring, is
allowed to cool to 40
oC. At a temperature of 38
oC to 40
oC, 2.02g of the mixture is filled into suitable, 2 ml plastic moulds. The suppositories
are allowed to cool to room temperature.
Example 13: Pessaries |
|
mg/pessary |
Active ingredient |
250 |
Anhydrate Dextrose |
380 |
Potato Starch |
363 |
Magnesium Stearate |
7 |
|
1000 |
[0088] The above ingredients are mixed directly and pessaries prepared by direct compression
of the resulting mixture.
Example 14: Antiviral and Toxicity Testing
[0089] Antiviral activity against the Human Immunodeficiency Virus (HIV) was determined
by measuring the ability of the compound to reverse the cytopathic effect of HIV infection.
This was determined by a quantitative assessment of cell growth monitored at the fifth
day post infection by a uptake test. Subconfluent (20-40,000 cells/well) human T lymphocyte
cell line MT4 cells infected with HIV were grown in 96-well microtiter dishes and
exposed to different dilutions of drug. After 5 days, the dye intake test was performed
on drug treated cultures and on HIV infected and mock infected MT4 cells. Under the
conditions of the test, HIV infection caused extensive cytopathic effect and prevented
cell growth by >80%. The antiviral effect of a drug is reported as an IC-50, i.e.
as the inhibitory concentration that would protect 50% of the cells from cell killing,
measured as 50% of that cell growth determined for uninfected MT4 cell controls.
[0090] Cell toxicity was assessed in a cell growth inhibition assay on uninfected MT4 cells
or on vero cells in a 96-well microtiter dish. Identical cell numbers of uninfected
cells were exposed to different dilutions of drug and cell viability determined daily
on replicate cultures using uptake of MTT. The concentration required for a 50% inhibition
of cell viability at 5 days is termed CCID-50.
Compound |
IC₅₀HIV |
CCID₅₀ |
2′,3′-dideoxy-5-ethynyl-3′-fluorouridine |
8.9µM |
224µM(MT4 cells) |
2′,3′-dideoxy-5-ethynyl-3′-fluorouridine |
- |
464µM(Vero cells) |